1 Isoflavones are an important group of secondary metabolites in accompany with soybean development. Due to their important roles for plant protection and human health, more and more attention has been paid on soybean isoflavones. In this study, the HPLC and Real-time PCR analyses were used to determine the isoflavone content and relative expression levels of genes encoding isoflavone synthesis relative enzymes during the seed development in two soybean cultivars with significantly different isoflavone content (cv. LHD2 and NHZ), and their correlation was also analyzed. We found that the isoflavone content increased gradually during the development of soybean seeds. In the meanwhile, the expression trends of different isoflavone synthesis relative enzyme genes differed significantly. The expression trends of CHS7, CHS8, CHR, CHI1A and IFS2 were consistent with isoflavone accumulation, while the expression trends of IFS1 and CHI1B1 were opposite to isoflavone accumulation. The expression trend of isoflavone reductase (IFR) differed between two cultivars. In LHD2, it was opposite to isoflavone accumulation, while in NHZ, it was consistent with isoflavone accumulation. Our results also demonstrated that different gene members in the same gene family exhibited different expression levels during the seed development. For instance, CHS7 and CHS8 showed higher expression levels than other members in chalcone synthase gene family, as well as the CHI1A in chalcone isomerase gene family. The expression level of IFS2 was also significantly higher than IFS1 in isoflavone synthase gene family. This result suggested functional differentiations among different members of the same gene family. Additionally, the correlation analysis between expression level and isoflavone accumulation showed that the correlations differed between two cultivars for different isoflavone synthesis relative enzyme genes. In LHD2, the expression levels of CHS7, CHS8 and IFS2 showed significant positive correlations with multiple isoflavone components, CHI1B1 exhibited significantly negative correlation with multiple isoflavone components. On the other hand, in NHZ, the expression level of IFR was significantly positively correlated with the accumulations of multiple isoflavone components. That implied a potential genetic basis for the varying isoflavone contents between both of soybean varieties. The correlation analysis among the expression levels of isoflavone biosynthesis relative enzyme genes during soybean seed development showed that PAL1 and CHS2 had significantly positive correlation with 4CL, and that CHS1 was significantly positively correlated with IFS2. This result suggested that these genes co-regulated the biosynthesis and accumulation of isoflavones. Taken together, our results provided substantial genetic basis on isoflavone biosynthesis and accumulation during soybean seed development, which will be useful to improve soybean isoflavones through genetic engineering in the future.